The invention relates to isolated peptides which are useful in treating stroke and other neurodegenerative diseases. The isolated peptides also are useful for binding calcium. The peptides preferably are conjugated to a compound which facilitates delivery across the blood-brain barrier.
Approximately 750,000 new strokes occur in the United States every year and cause about 250,000 deaths (Kittner et al., J. Am. Med. Assoc. 264:1267-1271, 1990). While the human suffering caused by stroke is enormous, both to the victims and their families, the economic costs are enormous as well. Long-term follow-up studies show that most stroke survivors experience permanent disability ranging from loss of vocational competence (71%), to requiring assistance with daily care (31%), to institutionalization (16%) (Gresham et al., N. Eng. J. Med. 293:954-959, 1975). Based on these data, roughly 300,000 persons permanently lose some function each year because of stroke.
The fundamental hypothesis in stroke research is that ischemia produces disability and death, not directly, but rather indirectly by initiating a cascade of cellular processes that eventually lead to neuronal death (Pulsinelli et al., Annals Neurol. 11:499-509, 1981; Choi, Trends Neurosci. 11:465-469, 1988). Until physicians can regenerate functional neurons to replace dead ones, the best hope for stroke victims is to intervene quickly with treatments that interrupt and reverse the cascade of events triggered by the primary ischemic event before they become irreversible.
The cascade of events begins about three to four minutes after ischemia: the first step is that the concentration of extracellular excitatory amino acids increases by 10- to 100-fold (Mayevsky, Brain Res. 524:1-9, 1990; Mitani and Katoaka, Neuroscience 42:661-670, 1991). These excitotoxic amino acids trigger a subsequent chain of events that includes calcium release from intracellular stores and eventually the expression of new genes. Dead neurons and irreversible loss of cognitive and behavioral function are results of this cascade which occurs hours after the initial ischemia.
A goal of anti-stroke treatment is to intervene in the cascade of neuronal death before it becomes irreversible, saving as many neurons as possible. A substantial body of work indicates that this theoretical possibility is a realistic goal. For example, several naturally occurring proteins can prevent neuronal death after excitotoxic damage in vitro or after experimental ischemia in vivo (Berlove et al., Soc. Neurosci. 17:1267, 1991; Shigeno et al., J. Neurosci. 11:2914-2919, 1991). These proteins (including nerve growth factor, brain derived neurotrophic factor, basic fibroblast growth factor, ciliary neurotrophic factor, and others) derive from two structurally related protein families, neurotrophins and cytokines, and are involved in the control of neuronal differentiation in the central and peripheral nervous system. The most likely mechanism by which these proteins protect neurons from ischemia seems to involve the expression of various genes. Presumably those gene products inhibit a cell death program which is triggered by the excitotoxins, and which could involve calcium release from intracellular stores. One of the most interesting previous findings shows that some of these neurotrophic factors can protect neurons from death when applied up to tens of minutes after the injury (Shigeno et al., 1991).
Other examples of compounds used to treat the neurodegenerative effects of cerebral ischemia include U.S. Pat. No. 5,559,095, which describes a method of treating ischemia-related neuronal damage using omega-conotoxin peptides and related peptides which bind to and block voltage-gated calcium channels, and U.S. Pat. No. 4,684,624, which describes treatment using certain opioid peptides. These peptides are not related to neurotrophins or cytokines.
While the neuroprotective effects of the neurotrophins are encouraging, their potential clinical application is limited by their large size (10 kD or greater) which prevents effective delivery through the blood-brain barrier (BBB). Neuroprotective molecules that can cross the BBB to act on neurons imperiled by cerebral ischemia will be more efficacious in the treatment of stroke. Molecules that protect neurons against the ischemic effects of stroke will also be useful for treating Alzheimer""s disease, as well as the memory deficits that are characteristic of the aging process.
It has now been discovered that peptides can be derived from a neurotrophin and maintain the neuroprotective capabilities of the larger protein. Peptides that maintain the neuroprotective effects of ependymin, a protein from which amino acid sequence of the peptides is partially derived, have been prepared. It has also been discovered that peptides which conform to the EF-hand rule of calcium binding proteins are neuroprotective.
According to one aspect of the invention, a composition comprising an isolated peptide is provided. The peptide includes the amino acid sequence set forth in SEQ ID NO:1. In certain embodiments, the isolated peptide includes the amino acid sequence of SEQ ID NO:2. In other embodiments, the isolated peptide binds calcium. In still other embodiments, the isolated peptide lacks one or more calcium coordination residues of the amino acid sequence of SEQ ID NO:1. Preferably, the foregoing isolated peptides include the amino acid sequence set forth in SEQ ID NO:3, and more preferably consists essentially of the amino acid sequence set forth in SEQ ID NO:3.
According to another aspect of the invention, a composition comprising an isolated peptide is provided. The isolated peptide includes the amino acid sequence set forth in SEQ ID NO:19 and in certain embodiments includes the amino acid sequence set forth in SEQ ID NO:10. In preferred embodiments, the isolated peptide includes the amino acid sequence set forth in any of SEQ ID Nos:11-18. Preferably the foregoing isolated peptides bind calcium.
In the foregoing compositions, the isolated peptide also can include 1-6 amino acids on one or more of the N-terminus and the C-terminus of the isolated peptide, wherein the amino acids are selected from the group consisting of lysine and arginine. In certain of the embodiments of these compositions, the isolated peptide comprises 2-4 lysines and/or arginines on the N-terminus or the C-terminus of the isolated peptide. In preferred embodiments, the isolated peptide is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:9, most preferably SEQ ID NO:4.
In the foregoing compositions, the isolated peptide also can include fatty acids. Preferred fatty acids include docosahexaenoic acid.
In certain embodiments of the foregoing compositions, the isolated peptide is non-hydrolyzable, which means that the peptide bonds are less readily hydrolyzed than peptide bonds formed between L-amino acids. Preferred non-hydrolyzable peptides include those selected from the group consisting of peptides comprising D-amino acids, peptides comprising a -psi[CH2NH]xe2x80x94 reduced amide peptide bond, peptides comprising a -psi[COCH2]xe2x80x94 ketomethylene peptide bond, peptides comprising a -psi[CH(CN)NH]xe2x80x94 (cyanomethylene)amino peptide bond, peptides comprising a -psi[CH2CH(OH)]xe2x80x94 hydroxyethylene peptide bond, peptides comprising a -psi[CH2O]xe2x80x94 peptide bond, and peptides comprising a -psi[CH2S]xe2x80x94 thiomethylene peptide bond. The most preferred isolated peptides are those which include 1-3 D-amino acids.
In the foregoing compositions, the isolated peptide is between 4 and 25 amino acids in length and preferably is between 10 and 20 amino acids in length.
In some embodiments of the invention, the isolated peptide is conjugated to a compound which facilitates transport across the blood-brain barrier into the brain. A blood brain barrier transport compound preferably is selected from the group consisting of docosohexaenoic acid, a transferrin receptor binding antibody, cationized albumin, Met-enkephalin, lipoidal forms of dihydropyridine, and cationized antibodies.
According to another aspect of the invention, a method for treating a subject having a condition characterized by cerebral ischemia is provided. The method includes administering to the subject an amount of an isolated peptide which includes the amino acid sequence of SEQ ID NO:1 effective to reduce the neurotoxic effect of cerebral ischemia in the subject. In certain embodiments, the isolated peptide is administered to the subject after the cerebral ischemia event. In other embodiments, the isolated peptide includes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 and SEQ ID NO:5. The isolated peptide also can be conjugated to a compound which facilitates transport across the blood-brain barrier into the brain, or the method can include administering a compound which increases transport across the blood-brain barrier.
In another aspect of the invention, a method for increasing neuronal cell AP-1 or NF-IL6 transcription factor activity in a subject is provided. The method includes administering to the subject an amount of an isolated peptide which includes the amino acid sequence of SEQ ID NO:1 effective to increase the activity of AP-1 or NF-IL6 in the subject. In some embodiments, the isolated peptide includes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 and SEQ ID NO:5. The isolated peptide also can be conjugated to a compound which facilitates transport across the blood-brain barrier into the brain, or the method can include administering a compound which increases transport across the blood-brain barrier.
According to still another aspect of the invention, a pharmaceutical composition is provided. The pharmaceutical composition includes an isolated peptide which comprises the amino acid sequence set forth in SEQ ID NO:1, and a pharmaceutically acceptable carrier. Preferably, the peptide reduces the neurotoxic effect of cerebral ischemia. The pharmaceutical composition also can include a compound which facilitates transport across the blood-brain barrier into the brain, which compound can be conjugated to the isolated peptide.
According to another aspect of the invention, a method for binding calcium is provided. The method includes contacting a calcium containing environment with one of the foregoing compositions, preferably a compostion which includes an isolated peptide which includes the amino acid sequence set forth in SEQ ID NO:10.
Another aspect of the invention provides a method for identifying a calcium-binding peptide. The method includes providing a putative calcium-binding peptide, contacting the putative calcium-binding peptide with an environment containing calcium, and determining the calcium binding of the peptide. In certain embodiments, the putative calcium binding peptide is a variant of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:19. In other embodiments, the step of providing a putative calcium-binding peptide includes providing a library having peptides including the amino acid sequences set forth in SEQ ID NO:1 and/or SEQ ID NO:19.
Another aspect of the invention provides a method for identifying a peptide which increases AP-1 or NF-IL6 transcription factor activity. The method includes the steps of providing a peptide, contacting the peptide with a cell which can express AP-1 or NF-IL6 transcription factor activity, and determining the AP-1 or NF-IL6 transcription factor activity to identify peptides which increase AP-1 or NF-IL6 transcription factor activity. In certain embodiments, the peptide is a variant of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:19. In other embodiments, the step of providing a peptide includes providing a library having peptides including the amino acid sequences set forth in SEQ ID NO:1 and/or SEQ ID NO:19.
According to another aspect of the invention, an isolated nucleic acid is provided. The nucleic acid encodes one of the foregoing isolated peptides. Also included in the invention are vectors, such as expression vectors, which include the isolated foregoing isolated nucleic acids.
The use of the foregoing compositions, isolated peptides and isolated nucleic acids in the preparation of medicament also in provided.
These and other aspects of the invention are described in greater detail below.